Project Summary/Abstract Elimination of misfolded proteins by ER-associated protein degradation (ERAD) ensures that proteins entering the secretory pathway are correctly folded and that ER stress is maintained at acceptably low levels. All ERAD pathways include a protein translocation process termed retrotranslocation, in which ubiquitinated ERAD substrates are selectively extracted from the ER before degradation by the cytosolic 26S proteasome. Despite its commonality in ERAD, many features of retrotranslocation have remained mysterious. We have recently made a major breakthrough in understanding retrotranslocation. By employing whole-genome yeast arrays, we have discovered the rhomboid family protein Dfm1 to be critical for the removal of membrane substrates, opening the door to a deep mechanistic understanding of retrotranslocation mechanisms and biology. Specifically, we w ill: 1) Determine the machinery and mechanisms involved in Dfm1-mediated retrotranslocation. 2) Characterize a novel retrotranslocation pathway induced in the absence of Dfm1. 3) Explore the new stress pathway that can arise in the absence of Dfm1. We will use a multifaceted approach including biochemistry, cell biology, genetics, functional genomics and proteomics to address these central questions in ERAD and membrane biology. We will leverage our unique in vivo and in vitro assays-and continue to devise new ones-to dissect the basic mechanisms of rhomboid-mediated retrotranslocation and its place in cell and organismal biology. A mechanistic understanding of retrotranslocation and the stress associated with its absence will establish foundational biological insights while unveiling therapeutic targets for a variety of critical pathways including protein misfolding, protein quality control, ER stress, and host-pathogen interactions.